The long range goal of this proposal is to characterize the functional roles of filamin, an important major component of smooth muscle and most other tissues. It is now clear that this protein is a major organizer of three dimensional actin filament arrays and it is likely to play a critical role in normal and diseased smooth muscle. Our long range goal will be approached through investigation of structural and functional properties of both the intact molecule and domain peptides. Functional sites on the filamin molecule will be more precisely located and characterized in greater detail. A major emphasis will involve the study of the regulation of filamin functions (self-association and actin binding) and factors that are likely to regulate these interactions. Possible changes in filamin organization, quantity and isoform distribution in several pathological states involving smooth muscle will also be investigated. For most studies, mild proteolysis will be used to divide this large multifunctional protein into smaller peptide units or domains. To facilitate the characterization of functional sites, a preliminary domain map of the molecule will be completed using conventional and HPLC peptide mapping. Functional properties of filamin will be investigated using established and novel methods for measuring protein - protein interactions in vitro. Pairwise protein associations and more complex multiprotein complexes will be analyzed. Attempts will be made to more precisely locate binding sites on the filamin molecule using several methods including: chemical crosslinking, site directed antibodies, and synthetic peptides. Biochemical and protein chemical methods will be used to search for factors that control and modulate known functional interactions. Possible regulatory factors that will be investigated include: phosphorylation and other posttranslational modifications, proteins or other factors whose interactions with filamin have not yet been identified, calpain cleavage, and ionic environment (especially Ca2+ levels). Also, biochemical methods and immunological probes developed in the above studies will be used to characterize filamin content, isoform expression and subcellular localization in normal and diseased smooth muscle.